PhytoCellTec™ Malus DomesticaPlant stem cells for skin stem cell protection

A Revolutionary Technology to Protect Skin Stem CellsPhytoCellTec™ Malus Domestica is a liposomal pre para-tion based on the stem cells of a rare Swiss apple.

Uttwiler Spätlauber is an endangered apple variety thatwas well-known for its excellent storability and thus itslongevity potential.

Mibelle Biochemistry has developed a novel technologyenabling the cultivation of rare and endangered specieslike Uttwiler Spätlauber. Thanks to this technology calledPhytoCellTec™, plant stem cells can be obtained andincorporated into cosmetic products to ensure thelongevity of skin cells.

Studies showed the double activity of PhytoCellTec™Malus Domestica:• it helps skin stem cells to maintain their characteristics

and their capacity to build new tissues• it delays aging and has an anti-wrinkle effect.

PhytoCellTec™ Malus Domestica is the first plant stemcell active ingredient on the market whose effect wasevaluated on human skin stem cells. This unique andrevolutionary ingredient is able to protect the mostprecious skin cells, the skin stem cells, against prematureaging.

Claim Ideas for PhytoCellTec™ Malus Domestica• Protects longevity of skin stem cells• Delays senescence of essential cells• Increases the vitality of skin stem cells• Combats chronological aging

Applications• Advanced “stem cell cosmetic” formulas• Real rejuvenation for face and body care• Innovative skin care formulations

Formulating with PhytoCellTec™ Malus Domestica• Recommended use level: 2 – 5 %• Incorporation: For cold processes, mix PhytoCellTec™

Malus Domestica into the aqueous phase. In cold / hotprocesses, add during the cooling phase below 40° C.

• Thermostability: Temperatures of up to 60° C for a shorttime do not affect the stability of PhytoCellTec™ MalusDomestica.

INCI / CTFA-DeclarationMalus Domestica Fruit Cell Culture Extract (and) XanthanGum (and) Glycerin (and) Lecithin (and) Phenoxyethanol(and) Aqua/ Water

Additional Information• PhytoCellTec™ Malus Domestica contains 9 %

of Malus Domestica stem cell extract• Available in a phenoxyethanol-free version• Available in powder form

PhytoCellTec™ Malus Domestica Plant stem cells for skin stem cell protection

May 2010

A Rare Apple with Incredible PropertiesUttwiler Spätlauber is a variety of a Swiss apple thatderives from a seedling planted in the middle of the 18th

century. It was very famous for its excellent storabilitywithout shriveling. Today apple cultivars are selected to maximize crop yield and sweet flavor. Thus UttwilerSpätlauber with its acid taste is now disappearing.

Uttwiler Spätlauber apples are rich in phytonutrients,proteins and long-living cells. They must have especiallylong-living tissue stem cells. Their particular compositionleads to incredible storability and longevity properties.

Stem Cells and LongevityLongevity is related to specific cells called stem cellswhich have a unique growth characteristic. These unspecialized (undifferentiated) cells can make identicalcopies of themselves as well as differentiate to becomespecialized cells.

Two basic types of stem cells are present in the humanbody: • embryonic stem cells found in blastocysts can grow

and differentiate into one of the more than 220 different cell types which make up the human body.

• adult stem cells located in some adult tissues can onlydifferentiate into their own or related cell types. These cells act as a repair system for the body but also maintain the normal turnover of regenerativeorgans such as blood, skin or intestinal tissues.

PhytoCellTec™ Malus DomesticaSwiss apple stem cells to protect skin stem cells

Stem Cells in the Human Skin In the human skin, two types of adult stem cells havebeen identified: • epithelial skin stem cells which are located in the

basal layer of the epidermis• hair bulge stem cells located in the hair follicle.

Fast regenerating tissues such as the epidermis areparticularly dependent on their stem cells because only stem cells retain the capacity to divide and thus to provide new tissues.

Epidermal stem cells have 2 main functions:• replenish and maintain the balance of cells within

the skin• regenerate damaged tissues.

Skin Stem Cells and Aging• Skin stem cells have a limited life expectancy.• Intrinsic or extrinsic stress factors can affect their

functionality.• With age, skin stem cells are less active and lower

in number.This explains why the depletion of skin stem cells isregarded as the primary cause of skin aging.

Plant Stem Cells to Protect Skin Stem CellsAll stem cells, independently of their origin (plant, animalor human) contain specific epigenetic factors whosefunction is to maintain the self-renewal capacity of stemcells. Therefore the Malus Domestica stem cells are usedto help to preserve the vitality of human skin stem cells.

Epidermal Stem Cells in the Human Skin

Stratum corneum

Differentiating progeny of one epidermal stem cell

Granular layer

Spinous layer

Basal layer

Dermis

Transient amplifying cell

Epidermal stem cell

Basement membrane

PhytoCellTec™ by Mibelle BiochemistryMibelle Biochemistry has developed a novel technologycalled PhytoCellTec™, enabling the large scale cultivationof cells from rare and endangered plant species.

This technology is based on the unique totipotency of plant cells that is to say:• the capacity of every plant cell to regenerate

new organs or even the whole plant • the capacity of every plant cell to dedifferentiate

and become a stem cell.

Our PhytoCellTec™ technology relies on the woundhealing mechanism of a plant: part of a plant is woundedto induce the formation of callus cells. This healing tissueconsists of dedifferentiated cells which are stem cells.Callus cells are harvested and cultivated in a suspension.A novel bioreactor system enables the large scale production.

To obtain the PhytoCellTec™ Malus Domestica cosmeticingredient, these stem cells are harvested and homoge-nized at 1200 bar together with phospholipids to encap-sulate and stabilize oil- and water-soluble componentsinto liposomes.

PhytoCellTec™ Malus Domestica is thus rich in epigeneticfactors and metabolites which assure the longevity of cellsand protect stem cells.

Advantages of PhytoCellTec™ TechnologyThis innovative technology developed by MibelleBiochemistry offers the following advantages:• possibility to cultivate cells of rare and endangered

plants based on small amounts of origin plant material• availability of plant material independent of the season

and market demand• plant material completely free of environmental

pollutants and pesticides• constant concentrations of metabolites in the

stem cells

PhytoCellTec™ Malus DomesticaAdvanced biotechnology to cultivate cells from rare plants

PhytoCellTecTM Malus Domestica Process

Disruption of the stem cells wall and encapsulation of their content into liposome Liposomal extract

Preparation of PhytoCellTec™ Malus Domestica

Wounding of plant material to induce callus formation▼

Harvesting of developed callus on solid media▼

Cultivation until complete dedifferentiation to obtain stem cells▼

Transfer of the stem cells into a suspension (liquid media)

Preparation of the Malus Domestica stem cells

▼

Preparation of Epidermal Stem Cells A novel Progenitor Cell Targeting technologywas used to prepare human epidermal stem cells.

This technology consists of culturing a skin sample in a medium specifically designed to mimic the micro-environment of the stem cell in the epidermis.

This special, fully defined cell culture medium leads to an enrichment of so called keratinocyte progenitor cellsthat can be considered as stem cells. This enrichment wasquantitatively controlled through FACS (Fluorescence-activated cell sorting) of cells which were labelled withCD34 and α6 integrin, two well-known markers of epider-mal stem cells. Compared to freshly isolated cells, thecell population of passage 3 was characterized by a 10-fold increase of CD34 /α6 integrin double labelled cells.

The Progenitor Cell Targeting technology provides a cellculture model which specifically enables the evaluation of the effects of compounds on epidermal stem cells.

The Capacity to Form a Colony is a Characteristic of Stem Cells Stem cells have the characteristic to form

colonies in vitro.

A colony consists of:• the progenitor cell• transient amplifying cells (cells in an intermediate state)• differentiated cells that have lost the capacity to divide.

The number of colonies formed is a value of the vitalprogenitor /stem cells and is called colony formingefficiency (CFE). To measure CFE, cells are seeded at a low density.

Enrichment of Keratinocyte Progenitor Cells

PhytoCellTec™ Malus Domestica Study results

Capacity to Form a Colony (CFE)

Progenitor cell (stem cell)

Transient amplifying cell

Differentiated cell

Freshly isolated cells Passage 3 in the specific medium

CD

34

α6 Integrin

6% doublelabelled

68% doublelabelled

S-234 / © Mibelle AG Biochemistry 2010

Increase of the Colony-Forming Efficiency (CFE)

Incr

ease

of t

he C

FE c

omp

ared

to

cont

rol i

n %

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0.01% 0.04 %

Concentration of Malus Domestica stem cell extract

0

20

40

60

80

100

PhytoCellTec™ Malus Domestica Study results

Maintenance of Epidermal Stem CellCharacteristicsThe epidermal stem cells obtained using the

previously described method, were treated with differentconcentrations of the Malus Domestica stem cell extract.Then, the effect of the Malus Domestica stem cell extracton the CFE was analysed and compared to a controlculture.

Results showed that the CFE was increased by 92 % in the presence of 0.04 % of the Malus Domestica stem cellextract.

Two other studies using this novel Progenitor CellTargeting technology also showed that:• this improvement of the CFE is not based on a general

growth stimulation of the progenitor cells• CFE is still improved when the Malus Domestica stem

cells are washed indicating that the stimulating effect is not due to the growth medium.

This clearly shows that Malus Domestica stem cell extracthelps the epidermal stem cells to maintain their stem cellcharacteristics (”stemness”).

S-234 / © Mibelle AG Biochemistry 2010

Keratinocyte Progenitor Cells in Culture

No cell growth

Maintenance of the Capacity to Build NewTissuesEpidermal stem cells were obtained using the

novel Progenitor Cell Targeting technology previouslydescribed. They were then cultured for different numbersof passages with and without 0.01 % Malus Domesticastem cell extract. Afterwards, their capacity to form a stratified epidermis was evaluated.

From the fresh epidermal stem cells, two kinds of cellswere tested:• “young” epidermal stem cells obtained from early

passage (passage 5)• “old” epidermal stem cells obtained from late

passage (passage 14).

The Malus Domestica stem cell extract was added to the culture from early passage.

Results showed that:• “old” epidermal stem cells lost their capacity to build

a 3D epidermis• but, if treated with Malus Domestica stem cell extract

from their early passage, “old” epidermal stem cellsretain their capacity to form a stratified epidermis. This 3D epidermis was made of all 4 epidermal layers(stratum basale, spinosum, granulosum, corneum) and was indistinguishable from the one obtained withthe “young” epidermal stem cells.

In presence of Malus Domestica stem cell extract, skinstem cells keep longer their ability to proliferate and to undergo the complex process of stratification anddifferentiation. Thus, the Malus Domestica stem cellextract helps the epidermal stem cells to retain theircapacity to build new tissues.

Maintenance of the Capacity to Form a 3D Epidermis

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Day 14 Day 21 Day 28

Young stem cells

Old stem cells

Old stem cells+ Malus Domestica

stem cell extract

PhytoCellTec™ Malus Domestica Study results

S-194 / © Mibelle AG Biochemistry 2010

Effect on Gene Expression in Senescent Dermal Fibroblasts Recently a cellular model for premature senes-

cence was established based on normal human dermalfibroblasts.

The fibroblasts were stressed with H2O2 in order toinduce premature senescence. The fibroblasts were then incubated with 2 % Malus Domestica stem cellextract. Then gene expression was analyzed with a cDNA array system comprising 150 probes.

Results showed that, in the control culture, several genes important for cellular proliferation and stimulationof cellular growth were down-regulated. But the MalusDomestica stem cell extract was found to neutralize this down-regulation and even induce an up-regulation of specific genes involved in the:• delay of senescence• protection against oxidative stress.

Age-Delaying Effect on Isolated Human Hair Follicles Human hair follicles in anagen phase were

isolated from skin fragments that are left after faceliftsurgery. Hair follicles are mini organs that represent anatural coculture model of epidermal and melanocytestem cells and differentiated cells. The follicles can bemaintained in a growth medium where they elongate until about day 14. Then, they gradually become senes-cent or undergo apoptosis due to the lack of bloodcirculation: they stop growing and start shrinking, this isthe necrosis process.

Isolated human hair follicles were incubated with MalusDomestica stem cell extract. Addition of 0.2 % of thisextract was found to slightly but clearly postpone senes-cence- and apoptosis-induced necrosis: follicles kept in the presence of the extract continued to elongate until day 18, whereas the control follicles started to shrink after day 14.

Increased Lifespan of Isolated Hair FolliclesReversion of Senescence Signs in Fibroblasts

Cha

nge

in le

ngth

com

par

ed t

o d

ay 1

4 in

%

Control 0.2% Malus Domestica stem cell extract

Day 16 Day 18 Day 20

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–70

–60

–50

–40

–30

–20

–10

0

10Genes after H2O2

Controlafter H2O2

+2 % MalusDomesticastem cellextract

cyclin B1: induces proliferation 73 130

cyclin E1: cell cycle regulator 78 135

p53: tumor suppressor gene 63 137

insulin-like growth factor II: cell proliferation enhancer 71 117

heme oxigenase 1: antioxidant enzyme 89 211

Gene, that is down-regulated by senescence induction (Control)

Gene, that is up-regulated or normalized in senescent cells by Malus Domestica stem cell extract

Anti-Wrinkle Effect on “Crow’s Feet” The anti-wrinkle effect of PhytoCellTec™ Malus Domestica was evaluated in a study

with 20 volunteers aged from 37 to 64.

An emulsion containing 2 % of PhytoCellTec™ MalusDomestica was applied twice daily for 28 days to thecrow’s feet. Wrinkle depth was determined by means of PRIMOS (phase-shifting rapid in vivo measurement of skin).

Results showed a significant and visible decrease in wrinkle depth for 100 % of the subjects.

Anti-Wrinkle Effect on Crow’s Feet

Before(day 0)

After(day 28)

2 % PhytoCellTec™Malus Domestica

S-186b / © Mibelle AG Biochemistry 2010

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Claim Ideas for PhytoCellTec™ Malus Domestica• Protects longevity of skin stem cells• Delays senescence of essential cells • Increases the vitality of skin stem cells• Combats chronological aging

Applications• Advanced “stem cell cosmetic” formulas• Real rejuvenation for face and body care• Innovative skin care formulations

Marketing Benefits• First plant stem cell active ingredient on the market (patent pending)• Proven efficacy on skin stem cells• Innovation in “stem cells” cosmetics• Breakthrough in anti-aging

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PhytoCellTec™ Malus DomesticaPlant stem cells for skin stem cell protection

The information contained in this publication is provided in good faith and is based on our current knowledge.No legally binding promise or warranty regarding the suitability of our products for any specific use is made.Claim ideas are offered solely for your consideration, investigation and verification. Mibelle Biochemistry will notassume any expressed or implied liability in connection with any use of this information.

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